Throughout this specification the use of the word “inventor” in singular form may be taken as reference to one (singular) inventor or more than one (plural) inventor of the present invention.
It is to be appreciated that any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the present invention. Further, the discussion throughout this specification comes about due to the realisation of the inventor and/or the identification of certain related art problems by the inventor. Moreover, any discussion of material such as documents, devices, acts or knowledge in this specification is included to explain the context of the invention in terms of the inventor's knowledge and experience and, accordingly, any such discussion should not be taken as an admission that any of the material forms part of the prior art base or the common general knowledge in the relevant art in Australia, or elsewhere, on or before the priority date of the disclosure and claims herein.
The technologies involved in and applied for cryopreserving of human and animal embryos are well established and with the application of suitable skill and know-how, the current technologies have achieved great improvement in the reliability and success for In Vitro Fertilisation procedures. For the purposes of this description, the following terms are taken to have the following definitions, with respect to the handling of embryos:
“Freezing” is the cooling of a liquid to a solid state which may include crystallisation.
“Vitrification” is the cooling of a liquid to a solid state without crystallisation.
“Cryopreservation” is a process where the cells are preserved by cooling to sub-zero temperature, typically −196 C.
“Thawing” is the process of changing from a frozen solid state to a liquid by gradual increase in temperature. This is most commonly associated with oocytes/embryos that have been cryopreserved by slow freezing techniques.
“Warming” is the process of changing from a vitrified solid state to a liquid state by rapid increases in temperature that prevents crystallisation. This is most commonly associates with oocytes/embryos that have been cryopreserved by vitrification techniques.
The techniques as understood and applied involve harvesting and cryopreservation of embryos, with a plurality of steps involving harvesting and extraction of oocytes, in vitro fertilisation thereof and the subsequent cryopreservation and storing of such fertilised eggs and the resultant embryos and/or late stage blastocysts. The multitude of steps and handling stages required are heavily reliant on a high level of know-how and skill via the technical operators. The embryos or blastocysts once frozen, are then made available as required and can be thawed and transferred to the recipient whereby successful implantation to the uterus can result in normal development of a foetus and a resultant pregnancy.
More recently, such cryopreservation techniques have been successfully applied to unfertilised eggs and oocytes. Oocyte cryopreservation involves harvesting, freezing and storing of eggs or oocytes from a donor female in an unfertilised state. Such frozen eggs can then be drawn from a storage bank, thawed and made available for fertilisation and transferred to a donor on demand.
The techniques of cryopreservation as applied to oocytes rather than fertilised eggs and embryos, has certain ethical and medical advantages and has been subject to increased research and experimentation to improve the techniques involved.
The process of cryopreservation, particularly when applied to “live” biological materials, involves a high degree of trauma for the biological material in question, particularly having regard to the multiple handling steps required in accordance with current techniques. In addition to the trauma experienced as a result of physical handling, the biological material is also subject to potential ice crystal formation during any freezing process, in addition to osmotic shock and toxic shock experienced during movement through a plurality of processing chemical solutions.
The traditional method of preparing frozen biological material includes the slow cooling of the material and its surrounding solution down to the storage temperature, with a view to deliberately initiating the formation of ice crystals remotely from the biological material per se. The traditional method is not optimal due to continuous formation of ice crystals. Alternative “vitrification” methods have been developed to address the ice crystal formation issues, however vitrification requires considerable technical skill for successful execution. Vitrification involves the transformation of the processing solution into a glass-like amorphous solid that is free from any crystalline structure, followed by extremely rapid cooling. The extremely rapid cooling is what enables the solution to achieve the glass-like amorphous state.
The application of either the traditional method of freezing or vitrification involves the use of chemical compounds and solutions, which are added to the biological material to minimise cell damage during the freezing processes. The chemical compounds and solutions are known as cryoprotectants and include permeating and non-permeating solutions. Permeating cryoprotectants are small molecules that readily permeate the membranes of the biological material with the formation of hydrogen bonds to the water molecules of the biological material with the aim of preventing ice crystallisation thereof. Examples of such permeating cryoprotectants are Ethylene Glycol (EG), Dimethyl Sulphoxide (DMSO) and Glycerol. At low concentrations in water, such permeating cryoprotectants lower the freezing temperature of the resultant solution and can assist in the prevention and minimisation of ice crystallisation. At higher concentrations which may differ at different cooling rates, such permeating cryoprotectants inhibit the formation of typical ice crystals and can lead to the development of a solid glass-like or vitrified state in which water is solidified prior to crystallisation or expansion. Toxicity of such permeating cryoprotectants increases with their increasing concentrations and is potentially toxic to the biological material in question and accordingly, the biological material must have minimal exposure to the permeating cryoprotectants over a very short period of time, or alternatively, exposure at a low temperature, whereby the metabolic rate of the biological material in question is reduced.
In contrast to the permeating cryoprotectants, the non-permeating cryoprotectants remain extracellular. Some examples of non-permeating cryoprotectants include disaccharides, trehalose and sucrose. The disaccharide cryoprotectants act by drawing free water from within the biological material and dehydrating the intracellular spaces. The resultant dehydration allows them to be used in combination with permeating cryoprotectants, such that the net concentration of the permeating cryoprotectant can be increased in the intracellular space. These techniques further assist the permeating cryoprotectant in preventing or minimising ice crystal formation.
During the vitrification process, permeating cryoprotectants may be added at a high concentration while the biological material's temperature is controlled at a predetermined level above freezing. However, because the toxicity of such high concentrations of permeating cryoprotectant can be substantial, it is not possible to retain the biological material at such temperatures for extended periods. Alternatively, a reduced time can be allowed for equilibrium after which the biological material, which may include oocytes or embryos are plunged directly into liquid nitrogen (where liquid nitrogen is hereinafter referred to as “LN2”) to effect freezing. The extremely rapid rate of cooling, minimises the negative effects of the cryoprotectant on the biological material and also, minimises ice crystal formation by encouraging vitrification.
The vitrification process involves exposing the biological material to a number of vitrification solutions. The vitrification solutions are typically added to successive wells in a multi-well culture dish, where the dish and solutions are warmed to a predetermined temperature, determined in accordance with the requirements of the biological material in question.
In a conventional protocol, the biological material is physically transferred to a first solution in a first well and then washed by physically moving the biological material or cell through the solution in question with a cell pipetting device. The washing process is repeated in a second, third and fourth well over predetermined periods of time until the biological material or cell is considered ready for cryopreservation. The biological material is then physically drawn up with a predetermined amount of vitrification solution using a pipette or other handling device. A droplet containing the biological material or cell to be vitrified is then pipetted onto the vitrification device. The vitrification device is then physically transferred with the droplet and biological material attached and directly plunged or sealed into a container that is plunged into LN2 or placed onto the surface of a vitrification block that has been pre-cooled with LN2. Once the biological material and the carrying fluid has become vitrified, the vitrification device is then inserted into a pre-chilled straw or other storage device, located in a slot in the vitrification block for subsequent transfer to long-term cold storage in either LN2 or LN2 vapour.
Various vitrification devices are used to manipulate the sample during the cryopreservation processes. Some propose a pipette style device in which the sample is sucked into a hollow tube which is then plunged directly into the solution or LN2. Such device is marketed by Irvine Scientific and sold as Cryotip®.
Other techniques use a loop/hook style device which will have a closed loop or an open hook made from plastic or metal wire attached to the end of a stem and is used to carry the biological sample. Such devices are marketed by Cryologic under the trade name of fibreplug™ or Cryoloop™ as defined in published international patent application WO00/21365.
Other tools are utilised as disclosed in international application WO 02/085110 “Cryotop” which is a flexible strip attached to a piece of plastic. In which the sample is placed on the strip and plunged directly into LN2.
Current prior art requires many embryo handling steps using multiple apparatus where every handling step increases the chance of losing the embryo. It is estimated that 1-2% of embryos lost are attributed to handling errors during the vitrification step.
The trauma associated with the previously described processes and in particular the trauma imposed by repeated physical handling and manipulation of extremely delicate biological material including eggs, cells, embryos and blastocysts, impacts on the survival rate and hence the success of the processes and methods previously described. Furthermore, the physical dynamics of a living embryo responding to osmolality changes introduce rapid shrinkage and expansion and other changes to the shape of the embryo which further challenge any handling, and in particular, automated handling of such biological materials. Any automation needs to manage such dynamics as well as manage a range of different embryo types, fluid movements along with a high range of fluid viscosities. Clearly, in order to maximise the chances of success and minimise trauma imposed on the materials being handled, it is highly desirable to reduce the physical handling of such delicate materials to an absolute minimum, which should mitigate cell shrinkage and expansion.
As noted above, the vitrification process involves exposing an embryo, or cell, to increasing concentrations of cryoprotectant solutions (also referred to as equilibration and vitrification solutions) so that water inside the cell is gradually removed and replaced. The concentrations of the fluids, the pace of fluid concentration changes that the cell experiences, the temperature at which the process takes place and the time over which it takes place are all important variables to achieve embryo viability in the end. Also important are the heat transfer rates, both the cooling during vitrification and warming to retrieve the embryo. Finally the addition of ‘warming’ solutions allows the cryoprotectants now inside the cell to be removed and replaced by water to ideally return the embryo to its initial state.
In addition to the above discussion there are a number of drawbacks with prior art, which can be summarised as follows: It is a very difficult and time consuming process which requires very skilled operator(s). Embryo loss is solely dependent on the skill of the operator. Variation in skills means variation in results in both embryo recovery (where an embryo is simply not found) and embryo survivability (embryo did not survive). Variation between lab environment, ie some labs might be running at 20° C. whilst others will be at 30° C. introduces problems. It is known that temperature variations or given temperature conditions can accelerate or decrease the biological reaction of the embryo. Over-exposing or under-exposing may damage the embryo. Variation in the processing time by humans means some embryos get over-exposed whilst others get under-exposed, ie overexposing the embryo in the final solution by 30 seconds may damage the embryo. Current consumables adapted for closed vitrification are heat sealed and therefore require cutting to retrieve the sample. The difficult and time consuming step of taking too long to retrieve the sample will damage the embryo ie more than 20 seconds. In practice moving embryos to increasing concentrations of cryoprotectant solutions is performed in a minimum amount of steps, usually 2-3, and this exposes cells to osmotic shock associated with considerable shrinking and subsequent expansion of cells, with the associated stress it causes on cell membranes and cytoskeleton.
Accordingly, variability may be one of the major issues with the current prior art systems. Vitrification variability can occur in the following areas:                Type of vitrification device being used. Currently there are over 15 types on the market.        The media being used. Currently there are over 10 media suppliers.        Embryologist skills and experience        Protocol (step time, temperature, cooling rate, warming rate, media volume)        Environment (temperature, humidity)        
Due to the variability in the environment, human involvement and protocols has greatly contributed to the lack of consistency in cryopreservation of biological material and the resultant low pregnancy rates.
It is therefore desirable to eliminate the variability by providing an automated system to control the environment and ensure a repeatable cryopreservation of biological materials.
There are 3 types of vitrification devices “closed” system, “semi closed” and “open” system. A “closed” system refers to a vitrification system that prevents direct contact between LN2 and the biological material. Cryotip® is considered to be a “closed” system. An “Open” system refers to a vitrification system that allows direct contact between LN2 and the biological material. Fibreplug™, Cryoloop™, and Cryotop® are all considered to be an “open” system. The problem with open systems is the direct contact with the requisite LN2 cooling solution with the risk of pathogen transmission to the biological sample at the time of freezing or during the storage. As the biological material is in contract with the LN2, contamination of sample can occur if the LN2 is contaminated or the LN2 can be contaminated if the sample is contaminated. Many countries have banned open systems due to the high risk of sample contamination.
Example of Cryotip® Protocol.
In the particular example of the Cryotip® system, there are a number of risky process steps that vary from low to medium to high risk in nature. For example, in the vitrification stages there is included the steps of introducing equilibration medium then vitrification medium then the loading and vitrification, which generally takes an estimated time of about 16 minutes. As a starting protocol for this stage embryos are transferred usually at a maximum of two at a time from culture dish to the equilibrium solution (ES) drop with a timer starting. Then for equilibrium media, the embryo is incubated undisturbed for about 6-10 minutes and 2 minutes prior to completion of this, four 20 μL drops of vitrification solution (VS1-4) are dispensed in a row. By the end of the equilibration time the embryos are transferred to a vitrification solution (VS), loaded, sealed and plunged within 90 seconds by transferring the embryos with minimal volume of medium from ES to VS1 for 5 seconds, then transfer to VS2 for 5 seconds then transfer to VS3 for 10 seconds. The high risk steps then occur with the loading and vitrification proper in which it is required to aseptically attach the wide end of a Cryotip® device to an aspiration tool, such as a luer syringe, using the Cryotip® connector. When the specimens are ready to load into the Cryotip® the metal cover sleeve is aseptically slid carefully along the straw to expose the fine tip end. The specimens are then gently loaded into the Cryotip® between its 2nd and 3rd mark by aspiration using the plunger on the syringe to control the uptake of medium and specimens being careful not to fill oocytes or embryos above the 3rd marker. Then the fine tip is heat sealed below the 1st mark then sliding the metal cover sleeve down over the fine tip to protect it. The connector and syringe are then removed and the wide end of the Cryotip® is heat sealed above the 4th mark. Finally the sealed Cryotip® is plunged with the metal covered side down first into the LN2 reservoir.